I. Field of the Invention
This invention is directed to tissue plasminogen activator (t-PA) variants having extended circulatory half-life as compared to wild-type human t-PA while substantially retaining its fibrin binding affinity. Certain variants additionally exhibit improved in vivo fibrinolytic potency. The invention also concerns methods and means for preparing these variants, and pharmaceutical compositions comprising them.
II. Description of Background and Related Art
Tissue plasminogen activator (t-PA) is a multidomain serine protease whose physiological role is to convert plasminogen to plasmin, and thus to initiate or accelerate the process of fibrinolysis.
Initial clinical interest in t-PA was raised because of its relatively high activity in the presence, as compared to the absence, of fibrin. Wild-type t-PA is a poor enzyme in the absence of fibrin, but the presence of fibrin strikingly enhances its ability to activate plasminogen. Without stimulation, the catalytic efficiency (catalytic rate constant (k.sub.cat) /Michaelis constant (K.sub.m)) of melanoma or recombinant human t-PA (Activase.RTM. t-PA) for the activation of plasminogen is approximately 1 nM.sup.-1 sec.sup.-1, whereas in the presence of fibrin or fibrin degradation products, this efficiency (pseudo-rate constant) is increased by several hundred-fold. This unusual biochemical property of t-PA is thought to translate clinically into a thrombolytic product that is less likely than non-fibrin-specific thrombolytics (such as streptokinase or urokinase) to induce systemic plasminogen activation [Sobel, B. E. et al., Circulation 69, 983-990 (1984)]. Recombinant human t-PA is used therapeutically as a fibrinolytic agent in the treatment of acute myocardial infarction and pulmonary embolism, both conditions usually resulting from an obstruction of a blood vessel by a fibrin-containing thrombus.
In addition to its striking fibrin specificity, t-PA exhibits several further distinguishing characteristics:
(a) T-PA differs from most serine proteases in that the single chain form of the molecule has appreciable enzymatic activity. Toward some small substrates, and toward plasminogen in the absence of fibrin, two chain t-PA has greater activity than one chain. In the presence of fibrin, however, the two forms of t-PA are equally active [Rijken et al., J. Biol. Chem. 257, 2920-5 (1982); Lijnen et al., Thromb. Haemost. 64, 61-8 (1990); Bennett et al., J. Biol. Chem. 266, 5191-5201 (1991)]. Most other serine proteases exist as zymogens and require proteolytic cleavage to a two-chain form to release full enzymatic activity.
(b) The action of t-PA in vivo and in vitro can be inhibited by a serpin, PAI-1 [Vaughan, D. E. et al., J. Clin. Invest 84, 586-591 (1989); Wiman, B. et al., J. Biol. Chem. 259, 3644-3647 (1984)].
(c) T-PA binds to fibrin in vitro with a K.sub.d in the .mu.M range.
(d) T-PA has a rapid in vivo clearance that is mediated by one or more receptors in the liver [Nilsson, S. et al., Thromb. Res. 39, 511-521 (1985); Bugelski, P. J. et al., Throm. Res. 53, 287-303 (1989); Morton, P. A. et al., J. Biol. Chem. 264, 7228-7235 (1989)].
A substantially pure form of t-PA was first produced from a natural source and tested for in vivo activity by Collen et al., U.S. Pat. No. 4,752,603 issued 21 Jun. 1988 (see also Rijken et al., J. Biol. Chem., 256:7035 [1981]). Pennica et al. (Nature, 301:214 [1983]) determined the DNA sequence of t-PA and deduced the amino acid sequence from this DNA sequence (see U.S. Pat. No. 4,766,075 issued 23 Aug. 1988).
Human wild-type t-PA has potential N-linked glycosylation sites at amino acid positions 117, 184, 218, and 448. Recombinant human t-PA (Activase.RTM. t-PA) produced by expression in CHO cells was reported to contain approximately 7% by weight of carbohydrate, consisting of a high-mannose oligosaccharide at position 117, and complex oligosaccharides at Asn-184 and Asn-448 [Vehar, G. A. et al., "Characterization Studies of Human Tissue Plasminogen Activator produced by Recombinant DNA Technology" Cold Spring Harbor Symposia on Quantitative Biology 1986; LI:551-562]. Position 218 has not been found to be glycosylated in native t-PA. Sites 117 and 448 appear to always be glycosylated, while site 184 is thought to be glycosylated in about fifty percent of the molecules. The t-PA molecules that are glycosylated at position 184 are termed Type II t-PA. and the molecules that are not glycosylated at position 184 are termed Type II t-PA. The most comprehensive analysis of the carbohydrate structures of CHO cell-derived human t-PA was carried out by Spellman et al., J. Biol. Chem. 264(24) 14100-14111 (1989), who showed that at least 17 different Asn-linked carbohydrate structures could be detected on the protein. These ranged from the high-mannose structures at position 117 to di-, tri- and tetra-antennary N-acetyllactosamine-type structures at positions 184 and 448. Type I and Type II t-PAs were reported to be N-glycosylated in an identical way at Asn-117 and Asn-448 positions, when isolated from the same cell line. For further details see also Parekh, Raj B. et al., Biochemistry 28, 7644-7662 (1989).
Analysis of the sequence of t-PA has identified the molecule as having five domains. Each domain has been defined with reference to homologous structural or functional regions in other proteins such as trypsin, chymotrypsin, plasminogen, prothrombin, fibronectin, and epidermal growth factor (EGF). These domains have been designated, starting at the N-terminus of the amino acid sequence of t-PA, as the finger (F) domain from amino acid 1 to about amino acid 44, the growth factor (G) domain from about amino acid 45 to about amino acid 91 (based on homology with EGF), the kringle-1 (K1) domain from about amino acid 92 to about amino acid 173, the kringle-2 (K2) domain from about amino acid 180 to about amino acid 261, and the serine protease (P) domain from about amino acid 264 to the carboxyl terminus at amino acid 527. These domains are situated essentially adjacent to each other, and are connected by short "linker" regions. These linker regions bring the total number of amino acids of the mature polypeptide to 527, although three additional residues (Gly-Ala-Arg) are occasionally found at the amino terminus. This additional tripeptide is generally thought to be the result of incomplete precursor processing, and it is not known to impart functionality. Native t-PA can be cleaved between position 275 and position 276 (located in the serine protease domain) to generate the 2-chain form of the molecule.
Each domain contributes in a different way to the overall biologically significant properties of the t-PA molecule. Domain deletion studies show that the loss of the finger, growth factor, or kringle-2 domains results in a lower affinity binding of the variant t-PA to fibrin [van Zonneveld, A. J. et al., Proc. Natl. Acad. Sci. USA 83, 4670-4677 (1986); Verheijen, J. H. et al., EMBO J. 5, 3525-30 (1986)], however, more recent results obtained with substitution mutants indicate that the kringle-2 domain is less involved in fibrin binding than earlier expected [Bennett, W. F. et al., J. Biol. Chem. 266 5191-5201 (1991)]. The domain deletion studies have implicated the finger and growth factor domains in clearance by the liver [Collen et al., Blood 71, 216-219 (1988); Kalyan et al., J. Biol. Chem. 263, 3971-3978 (1988); Fu et al., Thromb. es. 50, 33-41 (1988); Refino et al., Fibrinolysis 2,30 (1988); Larsen et al., Blood 73 1842-1850 (1989); Browne et al., J. Biol. Chem. 263, 1599-1602 (1988)]. The kringle-2 domain is responsible for binding to lysine. The serine protease domain is responsible for the enzymatic activity of t-PA and contains specific regions where mutations were shown to affect both fibrin binding and fibrin specificity (possibly direct interactions with fibrin), and other regions where only fibrin specificity is altered (possibly indirect interactions with fibrin) (Bennett et al., 1991,Supra). Studies with mutants resulting from site-directed alterations indicate the involvement of the glycosylation of t-PA in clearance [Lau et al., Bio/Technology 5, 953-958 (1987); Lau et al., Bio/Technology 6, 734 (1988)].
The relatively rapid clearance of wild-type human t-PA from the plasma, while it is an advantage in patients needing emergency intervention after thrombolysis, requires continuous intravenous administration to maintain therapeutic levels of t-PA in blood. The recommended total dose of Activase.RTM. t-PA for thrombolytic therapy in acute pulmonary embolism in adult patients is 100 mg given as a continuous intravenous infusion over 2 hours. T-PA derivatives with longer plasma half-life (slower clearance) could be administered as a bolus injection and would yield higher plasma concentrations than can be obtained with continuous infusion of wild-type t-PA, which may result in the reduction of the effective dose. Slower clearing t-PA mutants would offer particular advantages in the treatment of conditions such as deep vein thrombosis, treatment following reperfusion of an infarct victim, treatment of pulmonary embolism, or treatment of peripheral arterial thrombosis (peripheral vascular disease).
The need for t-PA variants that can be administered in a bolus form is underlined by recent interest in bolus administration of wild-type human t-PA (especially in single chain form) with the aim of promoting early infarct-related coronary artery patency and improving myocardial salvage [Verstraete et al., Lancet 14, 1566-1569 (1989) ; Neuhaus, JACC 14, 1566-1569 (1989) ; Khan et al., Am. J. Cardiol. 65, 1051-1056 (1990) ; Purvis, J. A. et al. Am. J. Cardiology 68, 1570-1574 (1991)]. Results of recent clinical studies indicate that bolus intravenous administration of wild-type human t-PA not only hastens the initiation of thrombolytic therapy but the rapid achievement of a relatively high t-PA concentration also enhances thrombolysis [Neuhaus, K. L., Supra, Topol, E. J., J. Am. Coll. Cardiol. 15, 922-924 (1990)].
T-PA variants with decreased clearance have been prepared by deleting individual amino acids, partial domains, or complete domains from the molecule. The following publications are representative of attempts to reduce the clearance rate of wild-type t-PA by deletion of part or all of the growth factor and/or finger domains, optionally combined with other mutations: Browne et al., 1988, Supra; Johannessen et al., Thromb. Haemostas, 63, 54-59 (1990); Collen et al., 1988, Supra; Kalyan et al., 1988, Supra; Sobel et al., Circulation 81, 1362-73 (1990); Cambier et al. J. Cardiovasc. Pharmacol., 11:468 (1988); Ann. Rev. Pharmacol. Toxicol., 30:91 (1990); Trill et al., Fibrinolysis 4, 131-140 (1990); U.S. Pat. No. 4,935,237 (issued 19 Jun. 1990); EP-A 241,208 (published 14 Oct. 1987); EP-A 240,334 (published 7 Oct. 1987). A t-PA variant with a duplicated kringle-2 domain, and reportedly reduced plasma clearance, was disclosed by Collen et al., Thromb. Haemost. 65, 174-180 (1991).
A variety of amino acid substitution t-PA variants have been evaluated for their ability to decrease the clearance rate and/or increase the half-life of t-PA. Substitutions in the amino acid regions 63-72 (and especially at positions 67 and 68), and 42-49 have been reported to increase the plasma half-life of wild-type human t-PA [see WO 89/12681, published 28 Dec. 1989 and Ahern et al., J. Biol. Chem. 265, 5540 (1990)]. The substitution of arginine at position 275 of native, mature t-PA with glutamic acid has been described to have a clearance rate about two times slower than that of wild-type human t-PA [Hotchkiss et al., Thromb. Haemost., 58:491 (1987)].
Another approach to decrease the clearance rate and/or extend the half-life of t-PA has been to complex the t-PA molecule with a second molecule. For example, a t-PA-polyethylene-glycol conjugate has been reported to reduce the rate of clearance of t-PA, as reported in EP-A 304,311 (published 22 Feb. 1989). A monoclonal antibody to t-PA has been reported to increase the functional half-life of t-PA in vivo without decreasing its activity (see EP-A 339,505 published 2 Nov. 1989).
The involvement of carbohydrates in the clearance of t-PA has also been studied. T-PA variants with different carbohydrate profile from that of wild-type human t-PA have been made and tested.
As examples of this approach, one or more of positions 60, 64, 65, 66, 67, 78, 79, 80, 81, 82, 103, 105, 107, and 250 have been substituted with appropriate amino acids to create molecules with glycosylation sites at or near some of these residues (see WO 89/11531, published 30 Nov. 1989 and U.S. Ser. No. 07/480691, filed 15 Feb. 1990, now abandoned, and its continuation U.S. Pat. No. 5,270,198 filed 21 Jan. 1992). Of these t-PA variants, the T103N extra-glycosylation t-PA mutant, for example, had about five fold slower clearance than native t-PA.
Other work focused on converting the glycosylation sites of wild-type t-PA to non-glycosylated sites. An unglycosylated variant of t-PA consisting of the kringle-2 and protease domains was described to have a slower plasma clearance than wild-type t-PA [Martin et al., Fibrinolysis 4 (Suppl. 3):9 (Abstract 26) (1990)]. Hotchkiss et al. [Thromb. Haemost., 60:255 (1988)] selectively removed oligosaccharide residues from the t-PA molecule, and demonstrated that the removal of these residues decreased the rate of clearance of t-PA. These researchers, and Lau et al. [(1987), Supra; (1988), Supra] also generated the t-PA variant N117Q (wherein asparagine at position 117 of wild-type human t-PA was substituted with glutamine) to prevent glycosylation at position 117. This variant, similarly to that obtained by enzymatic removal of the high mannose oligosaccharide at this position, exhibited an about two fold slower clearance rate than wild-type human t-PA. See also EP-A 238,304 published 23 Sep. 1987 and EP-A 227,462 published 1 Jul. 1987.
Further human t-PA glycosylation variants were described and reported to have reduced clearance rates in the following publications: Ahern et al., Supra (Q42N, H44E, N117Q t-PA); Collen et al., (1988), Supra [del(C6-I86)N117Q t-PA, and del (C6-I86)N117Q,N184Q,N448Q t-PA]; Haigwood et al., Prot. Engineer. 2, 611 (1989) (N117Q,N184Q t-PA).
We have found that the extension of circulating half-life of wild-type human t-PA by the addition of extra glycosylation at amino acid positions 103-105 was also accompanied by a loss in fibrin binding affinity and/or plasma clot lysis activity. For example, the replacement of threonine by asparagine at amino acid position 103 of wild-type human t-PA reduced the clearance rate about five fold, but also led to a loss in t-PA function in that the fibrin binding affinity and activity of t-PA were significantly reduced. As a result, although due to its lower clearance rate the plasma concentrations of this glycosylation variant were about 4-5 fold greater than for an equivalent dose of Activase.RTM. t-PA, because of its reduced activity, the improvement made in efficacy or in vivo fibrinolytic potency was little.
The present invention is based, among other things, upon specific successful research demonstrating that a loss in t-PA fibrin binding resulting from an alteration whose primary function is to improve the pharmacokinetic properties (reduce plasma clearance, extend circulatory half-life) of wild-type t-PA can be restored with an additional alteration without compromising the slower clearance rate or extended circulatory half-life achieved. The present invention is further based on experimental proof demonstrating that the in vivo clot lysis (fibrinolytic) potency of such t-PA variants can be significantly improved over that of wild-type human t-PA, in particular if the changes in the glycosylation pattern of t-PA are accompanied by additional specific alterations in the t-PA protease domain. The results are molecules with improved fibrinolytic potency with respect to wild-type t-PA, which are also capable of a more rapid lysis of plasma clots than wild-type t-PA and may additionally have improved fibrin-specificity.